CN112501113B

CN112501113B - HEK293 cell combined gradient culture medium and application thereof

Info

Publication number: CN112501113B
Application number: CN202110144051.1A
Authority: CN
Inventors: 肖志华
Original assignee: Shanghai Aopu Mai Biotechnology Co Ltd
Current assignee: Shanghai Aopu Mai Biotechnology Co Ltd
Priority date: 2021-02-03
Filing date: 2021-02-03
Publication date: 2021-04-27
Anticipated expiration: 2041-02-03
Also published as: CN112501113A

Abstract

A concentration gradient culture medium group for HEK293 cells comprises a basic culture medium, a feed culture medium X, a feed culture medium Y and a feed culture medium Z which are added in time sequence during culture; the concentrations of different components in the vitamins and the serum substitute are in different change trends from the basic culture medium to the supplemented culture medium Z, so that the effects of increasing the concentration of foreign proteins, improving the stability of cells and increasing the titer of packaged viruses are achieved.

Description

HEK293 cell combined gradient culture medium and application thereof

Technical Field

The invention relates to the technical field of biology, and particularly belongs to the field of HEK293 cell culture media.

Background

More and more genes and immunotherapy have been approved for clinical trials, and serum-free media has become the main cell culture medium for antibody and recombinant protein production. The culture medium is generally added at one time for cell culture, but some engineering cell types also need to bear the basic metabolism and the metabolism of artificially introduced exogenous substances, and the metabolism is related to the activation of a specific signal path, so that a plurality of substances are supplied, however, the addition of all the substances at one time and the direct reaching of the highest concentration can cause the osmotic pressure of the culture medium to be too high, the culture medium to be too viscous, the dissolved oxygen to be reduced, and the cell growth efficiency and the activity of the metabolites to be influenced. Fed batch can solve the above problems to some extent.

Similar studies have been made in the prior art, and EP592692B1 discloses a method for culturing 293 strain, in which a certain calcium ion concentration is maintained during initial cell culture, and when the cell is proliferated to about 3-fold cell density, sugar and calcium are added to the culture medium, so as to substantially inhibit the increase of cell density to maintain the production of protein, and the culture is stopped until the protein concentration is no longer significantly increased. However, in the above method, cells tend to aggregate into clumps by adding calcium salts, and although the increase in cell density is suppressed, in actual culture, the secretion of useful proteins is still suppressed to some extent and dissolved oxygen is affected, so that the growth state of cells is decreased; CN106754817A discloses a method for expressing recombinant ATX by batch culture of 293 cells, which utilizes FreeStyle293 expression culture medium for enlarged culture and feeds with different volumes at 24h, 48h and 60h, wherein the feed culture medium adopts the same components, and has no pertinence to different growth stages of the cells and no obvious improvement on the expression of foreign proteins; the CN102604889A method adopts a HEK293 cell line which is adaptive to serum-free culture, uses a culture medium with progressive concentration and serum content, but still adopts basically the same components, and does not carry out gradient optimization on specific nutrient components; the discovery of Yu et al is that in the 293 cell culture process, it is differentThe type of amino acid metabolism changes with time, glucose also has a certain important role in 293 culture and cannot be replaced by other components, and the growth rate of cells can be improved by adjusting the components of the culture medium and the number of cells, but the article only examines the amino acid variables and only carries out rough classification, and does not give clear culture medium components. The Leticia Liste-Calleja et al found that the cell density could be increased by using batch medium addition to culture 293 cells, but it was still limited to optimization of serum concentration and stepwise addition and cell densities up to 10⁶An order of magnitude.

According to the prior art, the HEK293 cell is an important platform for expression of antibodies and recombinant proteins, but in the culture process, the growth speed, density and state are often reduced due to cell strain difference, environmental difference and the like, so that the quality and yield of expressed drugs are limited. The addition of medium in batches improves this state, but the prior art techniques using batch culture still have certain problems. Therefore, optimizing the batch 293 cell culture medium is a problem to be solved urgently.

Disclosure of Invention

According to the problems existing in the prior art, the inventors want to optimize the 293 cell culture medium in batches in order to improve the activity of metabolites. Because serum may be an unknown contaminant, it has been gradually replaced by serum substitutes, typically including transferrin, lipid mixtures, insulin or IGF-1, growth factors, hormones, and the like; vitamins do not serve as an energy source in cell growth metabolism, but rather serve as a constituent of the cell. Vitamins, as bioactive substances that maintain cell growth, play a regulatory and controlling role in cell metabolism, and many vitamins participate in the components that constitute the active groups of various enzymes. The inventor finds that in the common components of the HEK293 cell culture medium, saccharides, inorganic salts and amino acid basic nutrients are added in batches, the influence on the cell culture state is small, and the content of trace elements is extremely low, so that the substances can be added at one time to maintain the normal cell culture state. The partial substances and vitamins of the serum substitute play a role in regulating and controlling cell metabolism due to participation in cell expression, the concentration and the type of the substances are closely related to a cell growth curve and a growth cycle, an adaptation process is adopted when cell culture is started, full-concentration nutrient components are directly added for partial components at the moment, the cell metabolism can be inhibited, the product activity is influenced, and the cell state is influenced when partial components are added at low concentration, so that the metabolic rate, the absorption selection, the concentration inhibition and the participation in a signal path are different due to different specific types in the growth and metabolism process of cells in the same type of substances, and the inventor uses the substance as a trigger to carry out the design of a secondary culture medium in batches. The concentration of the nutrient substances is classified by observing the metabolic rate, the cell growth state and the activity of the HEK293 cells on the nutrient substances, wherein amino acids, inorganic salts, sugars and trace elements can be added at one time without batch, and vitamins and partial serum substitutes need to be added in batches with different concentrations, and the concentrations in four groups of culture media, namely a basal culture medium, a feed culture medium X, a feed culture medium Y and a feed culture medium Z are classified into five grades of no concentration, low concentration, maintenance concentration, medium concentration and high concentration. Wherein the basic culture medium is a start culture medium, the feed culture medium X-feed culture medium Z belongs to a maintenance culture medium in general, and the metabolic activity of cells can be improved after the feed culture medium Z is added.

Class a (high-medium-dimensional): vitamin B1, vitamin B2, vitamin B12, vitamin C, vitamin E, niacinamide, insulin, transferrin;

class B (low-dimensional-medium-high): riboflavin, vitamin K2, vitamin K3, biotin, calcium D-pantothenate, cholesterol, bovine serum albumin, a lipid mixture;

class C (none-low-dimensional-medium): folic acid, inositol, pyridoxine hydrochloride, lipoic acid, choline chloride; polyethylene glycol, primidone.

The maintenance concentration is the concentration which is obtained by calculating the cell state and is only limited to the maintenance state through comprehensively considering the aspects of recording the cell density, observing the cell state, measuring the activity of a metabolic substance and the like, and the high concentration is the highest concentration for exciting the cell activity.

The invention discloses a culture medium group, the trend is divided into three categories, the category A is high concentration-medium concentration-maintaining concentration, and the related nutrient components comprise vitamin B1, vitamin B2, vitamin B12, vitamin C, vitamin E, nicotinamide, insulin and transferrin; the B is low concentration-maintaining concentration-medium concentration-high concentration, and the related nutritional ingredients comprise riboflavin, vitamin K2, vitamin K3, biotin, D-calcium pantothenate, cholesterol, bovine serum albumin, and lipid mixture; the C is non-low concentration-maintaining concentration-medium concentration, and the related nutritional ingredients comprise folic acid, inositol, pyridoxine hydrochloride, lipoic acid, choline chloride, polyethylene glycol and Primatone.

Wherein the exogenous protein is VEGF protein or M protein of Zika virus; the virus is a recombinant adenovirus expressing classical swine fever virus E2 protein.

The invention also discloses a culture method of the HEK293 cells, the HEK293 cells are recovered by using the basic culture medium disclosed by the invention, the HEK293 cells are replaced by a supplemented medium X after 12h, the supplemented medium Y is used for passage after continuous culture for 36h, and the supplemented medium Z is used for passage after continuous culture for 36h, wherein the inoculation density of the cells for each passage is 1-2 multiplied by 10⁶And/ml. The method comprises the following specific steps:

step 1: recovering the frozen HEK293 cells in a basal medium at 37 ℃ and 5% CO₂Resuscitating the cells in a basal medium of the culture medium set according to any of claims 1 to 5 to maintain the state of the cells;

step 2: counting the cells, observing the cell viability, when the recovery viability can reach more than 70 percent and/or the culture time reaches 12 hours, replacing the recovery viability with a supplemented medium X for adapting the cells to the culture environment, continuing to culture and calculate the cell viability, when the recovery viability is more than 95 percent and the cells are judged to be in the logarithmic phase and/or after the continuous culture time is 36 hours, starting to use the supplemented medium Y optimized by the invention, continuing to culture for 36 hours for subculture, using the supplemented medium Z to ensure that the cells are always in the logarithmic phase, and ensuring that the inoculation density is 1-2X 10⁶/ml；

And step 3: when the cell density reaches about 3-4X 10⁶When the cells are cultured at 3X 10⁶Inoculating the seed in six-well plate at a density of one mlCulturing overnight;

and 4, step 4: preparing 1-2mg/ml of transfection plasmid and 2-4mg/ml of transfection reagent PEI, diluting the transfection plasmid by using the optimized supplemented medium Z of the invention, uniformly mixing by using a vortex oscillator, adding into a six-hole plate, and supplementing the supplemented medium Z for culture;

and 5: 37 ℃ and 5% CO₂After incubation for 72h, digesting with 0.25% pancreatin, and centrifuging at 1500rpm/min to collect cells;

step 6: the cells and the expression products are harvested and the concentration of the protein of interest in the cell culture fluid is measured.

Optimizing the highest concentration of nutrient substances needing gradient addition according to the cell state: (Unit mg/L)

A type: vitamin B110, B21, B122, vitamin C10, vitamin E2, niacinamide 0.7; 20 of insulin; transferrin 20;

b type: riboflavin 0.5, vitamin K21, K31, biotin 0.01, calcium D-pantothenate 10; cholesterol 200; 100 parts of bovine serum albumin; lipid mixture 0.5% v/v;

class C: 0.02 parts of folic acid; 0.05 parts of inositol; pyridoxine hydrochloride 0.05; 0.5 parts of lipoic acid; 0.06 parts of choline chloride; polyethylene glycol 0.08 Primatone 5000.

And (4) forming four groups of culture media including a basic culture medium, a feed culture medium X, a feed culture medium Y and a feed culture medium Z according to the concentration optimization, and culturing HEK293 cells.

TABLE 1 gradient optimization medium group (unit: mg/L)

	Basic culture medium	Supplemented medium X	Supplemented medium Y	Supplemented medium Z
					Amino acids
Arginine	100-300	100-300	100-300	100-300
					Alanine	0-30	0-30	0-30	0-30
Glutamic acid	100-200	100-200	100-200	100-200
					Asparagine	50-200	50-200	50-200	50-200
Aspartic acid	50-250	50-250	50-250	50-250
					Cystine	10-500	10-500	10-500	10-500
Cysteine	100-200	100-200	100-200	100-200
					Glycine	50-100	50-100	50-100	50-100
Histidine	300-600	300-600	300-600	300-600
					Isoleucine	100-500	100-500	100-500	100-500
Leucine	800-1200	800-1200	800-1200	800-1200
					Lysine	700-1500	700-1500	700-1500	700-1500
Methionine	100-300	100-300	100-300	100-300
					Phenylalanine	100-300	100-300	100-300	100-300
Proline	0-50	0-50	0-50	0-50
					Serine	100-500	100-500	100-500	100-500
Threonine	100-300	100-300	100-300	100-300
					Glutamine	100-8000	100-8000	100-8000	100-8000
Valine	10-500	10-500	10-500	10-500
					Tyrosine	100-800	100-800	100-800	100-800
Tryptophan	50-150	50-150	50-150	50-150
					Inorganic salt
MgSO₄	30-60	30-60	30-60	30-60
					MgCl₂	50-100	50-100	50-100	50-100
NaCl	100-200	100-200	100-200	100-200
					CaCl₂	10-200	10-200	10-200	10-200
Na₂HPO₄.2H₂O	50-100	50-100	50-100	50-100
					NaH₂PO₄.2H₂O	50-100	50-100	50-100	50-100
C₃H₃NaO₃	10-50	10-50	10-50	10-50
					Trace elements
CuSO₄.5H₂O	0.001-0.01	0.001-0.01	0.001-0.01	0.001-0.01
					AlCl₃	0.005-0.05	0.005-0.05	0.005-0.05	0.005-0.05
CoCl₂.6H₂O	0.001-0.01	0.001-0.01	0.001-0.01	0.001-0.01
					Na₂SeO₃	0.001-0.05	0.001-0.05	0.001-0.05	0.001-0.05
NiCl₂	0.001-0.01	0.001-0.01	0.001-0.01	0.001-0.01
					MnCl₂	0.005-0.5	0.005-0.5	0.005-0.5	0.005-0.5
ZnSO₄.7H₂O	0.005-0.05	0.005-0.05	0.005-0.05	0.005-0.05
					FeSO₄.7H₂O	0.005-0.05	0.005-0.05	0.005-0.05	0.005-0.05
Candy
					Glucose	1000-5000	1000-5000	1000-5000	1000-5000
Glucan	1-100	1-100	1-100	1-100
					Vitamin preparation
Vitamin B1	9.8-10.2	5.5-9.8	4.5-5.5	4.5-5.5
					Vitamin B2	0.8-1.2	0.52-0.8	0.48-0.52	0.48-0.52
Vitamin B12	1.8-2.2	1.2-1.8	0.8-1.2	0.8-1.2
					Vitamin C	9.8-10.2	5.2-9.8	4.8-5.2	4.8-5.2
Vitamin E	1.8-2.2	1.2-1.8	0.8-1.2	0.8-1.2
					Nicotinamide	0.65-0.75	0.37-0.65	0.33-0.37	0.33-0.37
Riboflavin	0.1-0.2	0.2-0.3	0.3-0.48	0.48-0.52
					Vitamin K2	0.1-0.3	0.3-0.6	0.6-0.8	0.8-1.2
Vitamin K3	0.1-0.3	0.3-0.6	0.6-0.8	0.8-1.2
					Biotin	0.001-0.003	0.003-0.006	0.006-0.008	0.008-0.012
D-calcium pantothenate	2-3	3-6	6-9	9-11
					Folic acid	0	0.003-0.006	0.008-0.015	0.018-0.022
Inositol	0	0.015-0.02	0.02-0.03	0.048-0.052
					Pyridoxine hydrochloride	0	0.012-0.02	0.02-0.03	0.048-0.052
Lipoic acid	0	0.12-0.2	0.2-0.4	0.48-0.52
					Choline chloride	0	0.014-0.02	0.02-0.05	0.055-0.065
Serum replacement
					Insulin	18-22	12-18	9-11	9-11
Transferrin	18-22	12-18	9-11	9-11
					Cholesterol	47-53	90-110	110-190	190-210
Bovine serum albumin	34-36	45-55	55-95	95-105
					Lipid mixture	0	0.1-0.2%v/v	0.2-0.4%v/v	0.4-0.6%v/v
Primatone	0	1700-1900	1900-4800	4800-5200
					Polyethylene glycol	0	0.03-0.04	0.04-0.07	0.07-0.09

Table 2 lipid mixture composition: (Unit: mg/L)

Oleic acid	5-20
		Linoleic acid	5-20
Linolenic acid	5-20
		Palmitic acid	5-20
Twain (T)	80-2000
		FluronicF-68	1100000-1300000
Myristic acid	5-20
		Stearic acid	5-20
DL-acetic acid-tocopherol	70-80
		Ethanolamine	10-25

Advantageous effects

In the prior art, although a technical scheme of adding culture media in a gradient manner for domestication exists, the culture media are generally based on serum concentration change and generally gradually increase. The inventors have found that different components of the vitamin and serum replacement have different effects on the activity of the cells at different stages of cell growth, and that not all components increase in stages to favor cell growth. Different components in the vitamin and serum substitute are optimized, five gradients of high concentration, medium concentration, maintenance concentration, low concentration and no concentration are separated, and the five gradients appear in 4 sets of culture media including a basic culture medium and a feeding culture medium X-3 according to different concentration change trends, so that a cell signal path is fully activated, and the technical effects of improving the cell stability, improving the activity of metabolic protein and packaging the virus titer are realized.

Drawings

FIG. 1 protein concentration (in mg/L) was determined by in vitro protein expression.

FIG. 2 photograph of morphology of HEK293 cells.

FIG. 3 HEK293 cells used for virus production in 20 generations of culture (unit: hour).

FIG. 4 HEK293 cells Virus titre (units-lgTCID) cultured 20 passages₅₀）。

FIG. 5 cell culture interval optimization.

FIG. 6 cell seeding density optimization.

Detailed Description

Example 1 Combined gradient Medium-high concentration Medium-commercial Medium configuration

1) Configuration of gradient-adding combined culture medium

TABLE 3 gradient addition Combined Medium formulation (unit: mg/L)

	Basic culture medium	Supplemented medium X	Supplemented medium Y	Supplemented medium Z
					Amino acids
Arginine	125	125	125	125
					Alanine	12.5	12.5	12.5	12.5
Glutamic acid	135.8	135.8	135.8	135.8
					Asparagine	186.7	186.7	186.7	186.7
Aspartic acid	128.5	128.5	128.5	128.5
					Cystine	386	386	386	386
Cysteine	118	118	118	118
					Glycine	78.5	78.5	78.5	78.5
Histidine	301	301	301	301
					Isoleucine	267.1	267.1	267.1	267.1
Leucine	287	287	287	287
					Lysine	232.5	232.5	232.5	232.5
Methionine	132	132	132	132
					Phenylalanine	108	108	108	108
Proline	17	17	17	17
					Serine	101.5	101.5	101.5	101.5
Threonine	108	108	108	108
					Glutamine	489	489	489	489
Valine	98	98	98	98
					Tyrosine	109	109	109	109
Tryptophan	52	52	52	52
					Inorganic salt
MgSO₄	47.5	47.5	47.5	47.5
					MgCl₂	57.2	57.2	57.2	57.2
NaCl	183	183	183	183
					CaCl₂	15	15	15	15
Na₂HPO₄.2H₂O	78	78	78	78
					NaH₂PO₄.2H₂O	72	72	72	72
C₃H₃NaO₃	22.5	22.5	22.5	22.5
					Trace elements
CuSO₄.5H₂O	0.01	0.01	0.01	0.01
					AlCl₃	0.02	0.02	0.02	0.02
CoCl₂.6H₂O	0.005	0.005	0.005	0.005
					Na₂SeO₃	0.028	0.028	0.028	0.028
NiCl₂	0.035	0.035	0.035	0.035
					MnCl₂	0.28	0.28	0.28	0.28
ZnSO₄.7H₂O	0.013	0.013	0.013	0.013
					FeSO₄.7H₂O	0.038	0.038	0.038	0.038
Candy
					Glucose	5000	5000	5000	5000
Glucan	80	80	80	80
					Vitamin preparation
Vitamin B1	10	8	5	5
					Vitamin B2	1	0.8	0.5	0.5
Vitamin B12	2	1.6	1	1
					Vitamin C	10	8.5	5	5
Vitamin E	2	1.5	1	1
					Nicotinamide	0.7	0.65	0.35	0.35
Riboflavin	0.2	0.3	0.32	0.5
					Vitamin K2	0.2	0.5	0.78	1
Vitamin K3	0.2	0.5	0.7	1
					Biotin	0.003	0.005	0.007	0.01
D-calcium pantothenate	3	5	8.3	10
					Folic acid	0	0.003	0.01	0.02
Inositol	0	0.015	0.025	0.05
					Pyridoxine hydrochloride	0	0.018	0.025	0.05
Lipoic acid	0	0.12	0.25	0.5
					Choline chloride	0	0.014	0.03	0.06
Serum replacement
					Insulin	20	17	10	10
Transferrin	20	18	10	10
					Cholesterol	50	100	150	200
Bovine serum albumin	35	50	75	100
					Lipid mixture	0	0.15%v/v	0.25%v/v	0.5%v/v
Primatone	0	1800	2500	5000
					Polyethylene glycol	0	0.032	0.04	0.08

Table 4 lipid mixture composition: (Unit: mg/L)

Oleic acid	15
		Linoleic acid	15
Linolenic acid	15
		Palmitic acid	15
Twain (T)	1800
		FluronicF-68	1200000
Myristic acid	15
		Stearic acid	15
DL-acetic acid-tocopherol	75
		Ethanolamine	12

2) Commercial GIBCO medium was purchased as group B.

3) The medium was prepared according to the following formula, i.e., the concentration was high, and the medium was added at one time during the culture, and the fluid supplement was the same-i.e., control group C, wherein the formulation of the lipid mixture was the same as in Table 4.

TABLE 5 high concentration Medium C group (unit: mg/L)

Example 2: exogenous protein expression and virus packaging experiment of combined culture medium

1) In vitro expression of VEGF foreign proteins

Step 1: the frozen HEK293 cells were thawed in basal medium (cells purchased from GIBCO Co.), and the thawing conditions wereAt 37 ℃ with 5% CO₂(ii) a Step 2: counting the cells, observing the cell viability, when the recovery viability can reach more than 70 percent and/or the culture time reaches 12 hours, replacing the recovery viability with a supplemented medium X for adapting the cells to the culture environment, continuing to culture and calculate the cell viability, when the recovery viability is more than 95 percent and the cells are judged to be in the logarithmic phase and/or after the continuous culture time is 36 hours, starting to use the supplemented medium Y optimized by the invention, continuing to culture for 36 hours for subculture, using the supplemented medium Z to ensure that the cells are always in the logarithmic phase, and ensuring that the inoculation density is 1-2X 10⁶/ml；

And step 3: when the cell density reaches about 3-4X 10⁶When the cells are cultured at 3X 10⁶Inoculating the culture solution into a six-well plate at a density of/ml for overnight culture;

and 4, step 4: preparing 1-2mg/ml of transfection plasmid and 2-4mg/ml of transfection reagent PEI, diluting the transfection plasmid by using the optimized supplemented medium Z of the invention, uniformly mixing by using a vortex oscillator, adding into a six-hole plate, and supplementing the supplemented medium Z for culturing;

Comparative example 1:

the procedure was the same as above, using GIBCO commercial medium-group B medium throughout the procedure.

Comparative example 2:

the steps are the same as above, and a control group high-concentration culture medium-group C culture medium is adopted in the whole process.

2) In vitro expression of Zika virus antigen M protein

The procedure was as in the above example, and the Zika virus antigen M protein was expressed using the recombinant shuttle plasmid pShuttle2-CMV-JE-prM-E (construction method see CN111088271A which construction method is herein incorporated by reference in its entirety).

Comparative example 1:

Comparative example 2:

3) In vitro expression of foreign proteins containing the extracellular domain of IL-17

The procedure was the same as in the above example, using the pME18X vector, introducing the foreign gene, constructing the plasmid pME18X-N-FLAG ILT7 expressing the desired protein, expressing the foreign protein containing the extracellular domain of IL-17 (see CN110776566A for construction methods, which are again incorporated by reference in their entirety).

Comparative example 1:

Comparative example 2:

TABLE 6 in vitro protein expression assay protein concentration (unit: mg/L)

	VEGF	Zika virus M protein	Exogenous proteins comprising the extracellular domain of IL-17
				Group A-gradient Medium	1085	795	409
Group B-GIBCO Medium	803	589	352
				Group C-high concentration Medium	586	605	308

The results are shown in FIG. 1, and it can be seen that the gradient culture medium can obviously improve the activity of different kinds of foreign proteins.

4) For packaging adenovirus

HEK293 cell culture method is the same as 1), when the cell is added into a six-well plate, the recombinant adenovirus rADV-SFV-E2 is inoculated, and the titer is 10⁴TCID₅₀ ^/ml, continuously culturing for 46h, harvesting cells, fixing with 4% formaldehyde for 10-30min, treating with 0.1-0.3TritonX100 for 10min, adding anti-CSFV E2 protein monoclonal antibody HQ05, incubating at room temperature for 1h, adding FITC-labeled goat anti-mouse IgG (1: 100), observing cell morphology under a fluorescence microscope, and calculating virus titer by limiting dilution.

Results

1 morphology of

The morphology and the growth state of the cells are observed by using a microscope after the optimized culture medium is added, the growth state of the cells is good before the supplement culture medium X is used, the growth speed of the cells is accelerated after the supplement culture medium X, Y is used, the cells gradually show a suspension state after average passage for about 72 hours, and the cells show cell masses during static culture, so that the method can be adapted to the optimized cell culture medium.

The results are shown in FIG. 2, the left panel is a fast growth diagram, and the right panel is a cytopathic state in suspension.

2 cell stability

The cells were serially passaged 20 times, and the virus titer was measured in each passage according to the above method, during which time required for counting lesions and the virus titer in each passage were counted, and the average value was calculated. The results show that the group A gradient culture medium has the advantages of average lesion time, average virus titer, good stability, relatively stable trend of 20 generations and lower standard deviation than the group B and the group C; the gradient culture medium used by the invention can improve the adenovirus proliferation capability of cells, and the virus production time is averagely reduced by 10.2% compared with that of a B group-GIBCO commercial culture medium, and is averagely reduced by 13.4% compared with that of a C group-high concentration culture medium; the virus titer is improved by 73.7 percent compared with the group B-GIBCO commercial culture medium and 118 percent compared with the group C-high concentration culture medium. The results are shown in tables 7-9, FIGS. 3-4.

TABLE 7 HEK293 cells for 20 generations of virus production (unit: h)

	Group A	Group B	Group C
				F1	101.9	109	118.2
F2	99.2	107.3	110.2
				F3	94.6	109.3	102.5
F4	102.3	105.8	120.8
				F5	96.2	115.5	118.6
F6	99.5	99.9	107.6
				F7	97	100.3	119.3
F8	101.1	109.2	109.6
				F9	97.8	102.6	111.6
F10	102.4	106.5	106.7
				F11	95.1	100.2	119.3
F12	96.8	116.3	108.4
				F13	95.8	115.2	113.9
F14	96.2	107.6	104.6
				F15	99.9	111.3	118.3
F16	95.9	109.2	106.7
				F17	97	106.7	109.7
F18	94.9	110.9	116.7
				F19	95.6	109.8	116.9
F20	99.8	110.7	119.6

TABLE 8 HEK293 cells cultured for 20 passages Virus titer (unit-lgTCID)₅₀）

	Group A	Group B	Group C
				F1	8.15	7.67	7.25
F2	7.95	7.85	7.55
				F3	8	7.56	7.29
F4	8.01	7.74	7.99
				F5	8.28	7.95	7.65
F6	7.98	8.01	8.08
				F7	8.03	8.03	7.65
F8	8.44	7.68	7.46
				F9	8.03	8.04	8.01
F10	7.88	7.56	7.48
				F11	8.05	8.01	8.12
F12	8.32	8.03	8.12
				F13	8.01	7.59	7.86
F14	8.22	7.66	8.01
				F15	7.99	7.98	8.03
F16	8.03	8.04	7.65
				F17	7.99	7.98	7.66
F18	8.02	7.65	7.95
				F19	8.11	7.96	7.35
F20	7.89	7.44	7.41

TABLE 9 mean lesion time and mean viral titer of HEK293 cells after 20 passages

	Mean time to lesion (h)	Mean viral Titer (TCID)₅₀/ml）
			Group A-gradient Medium	97.95±4.05	10^8.06±0.38
Group B-GIBCO Medium	108.17±9.10	10^7.82±0.22
			Group C-high concentration Medium	112.96±10.80	10^7.72±0.40

Example 3 culture Condition optimization of Combined gradient Medium

In-vitro expression VEGF protein is taken as a judgment standard to optimize 293 cell culture parameters

1) Culture time interval optimization

Reference example 2 VEGF protein expression procedure,

TABLE 10 cultivation time Interval optimization

	Adding basic culture medium	Adding supplemented medium X	Adding supplemented medium Y	Adding a supplementary medium Z
					Comparative example 1	Cell resuscitation	10h	24h	24h
Comparative example 2	Cell resuscitation	12h	36h	36h
					Comparative example 3	Cell resuscitation	16h	48h	48h
Comparative example 4	Cell resuscitation	24h	72h	72h

The results are shown in fig. 5, and it can be seen that comparative example 2 with recovery time of 12h and subsequent stable passage time of 2.5 days has higher protein concentration, and the inventors have also tested other foreign proteins with substantially the same results, and the data are omitted here.

2) Cell density

The subculture cell seeding density was optimized and different cell seeding densities were selected for seeding at a specific density after cell counting at the time of subculture, using the above comparative example 2.

TABLE 11 cell Density optimization

Density of inoculation	Concentration of VEGF protein
		0.5×10⁶/ml	988
1×10⁶/ml	1069
		1.5×10⁶/ml	1073
2×10⁶/ml	1070
		2.5×10⁶/ml	975
3×10⁶/ml	889

The results show that 1-2X 10⁶The/ml results are not significantly different and are lower than 1X 10⁶A sum of/ml and greater than 2X 10⁶Per ml, therefore 1-2X 10 are used⁶The cell seeding density per ml may be favorable for foreign protein expression.

Claims

1. A culture medium group comprises a basic culture medium, a supplementary culture medium X, a supplementary culture medium Y and a supplementary culture medium Z which are added according to the cell culture time sequence, wherein the components of the culture medium group comprise basic components of amino acid, inorganic salt, trace elements, sugar, vitamin and serum substitute, and the culture medium group is characterized in that the concentration of each component in the vitamin and serum substitute is as follows by taking mg/L as a unit:

vitamin preparation Basic culture medium Supplemented medium X Supplemented medium Y Supplemented medium Z Vitamin B1 9.8-10.2 5.5-9.8 4.5-5.5 4.5-5.5 Vitamin B2 0.8-1.2 0.52-0.8 0.48-0.52 0.48-0.52 Vitamin B12 1.8-2.2 1.2-1.8 0.8-1.2 0.8-1.2 Vitamin C 9.8-10.2 5.2-9.8 4.8-5.2 4.8-5.2 Vitamin E 1.8-2.2 1.2-1.8 0.8-1.2 0.8-1.2 Nicotinamide 0.65-0.75 0.37-0.65 0.33-0.37 0.33-0.37 Riboflavin 0.1-0.2 0.2-0.3 0.3-0.48 0.48-0.52 Vitamin K2 0.1-0.3 0.3-0.6 0.6-0.8 0.8-1.2 Vitamin K3 0.1-0.3 0.3-0.6 0.6-0.8 0.8-1.2 Biotin 0.001-0.003 0.003-0.006 0.006-0.008 0.008-0.012 D-calcium pantothenate 2-3 3-6 6-9 9-11 Folic acid 0 0.003-0.006 0.008-0.015 0.018-0.022 Inositol 0 0.015-0.02 0.02-0.03 0.048-0.052 Pyridoxine hydrochloride 0 0.012-0.02 0.02-0.03 0.048-0.052 Lipoic acid 0 0.12-0.2 0.2-0.4 0.48-0.52 Choline chloride 0 0.014-0.02 0.02-0.05 0.055-0.065 Serum replacement Basic culture medium Supplemented medium X Supplemented medium Y Supplemented medium Z Insulin 18-22 12-18 9-11 9-11 Transferrin 18-22 12-18 9-11 9-11 Cholesterol 47-53 90-110 110-190 190-210 Bovine serum albumin 34-36 45-55 55-95 95-105 Lipid mixture 0 0.1-0.2%v/v 0.2-0.4%v/v 0.4-0.6%v/v Primatone 0 1700-1900 1900-4800 4800-5200 Polyethylene glycol 0 0.03-0.04 0.04-0.07 0.07-0.09

。

2. The media set of claim 1, wherein the vitamin and serum replacement formula in the media set is, in mg/L:

vitamin preparation Basic culture medium Supplemented medium X Supplemented medium Y Supplemented medium Z Vitamin B1 10 8 5 5 Vitamin B2 1 0.8 0.5 0.5 Vitamin B12 2 1.6 1 1 Vitamin C 10 8.5 5 5 Vitamin E 2 1.5 1 1 Nicotinamide 0.7 0.65 0.35 0.35 Riboflavin 0.2 0.3 0.32 0.5 Vitamin K2 0.2 0.5 0.78 1 Vitamin K3 0.2 0.5 0.7 1 Biotin 0.003 0.005 0.007 0.01 D-calcium pantothenate 3 5 8.3 10 Folic acid 0 0.003 0.01 0.02 Inositol 0 0.015 0.025 0.05 Pyridoxine hydrochloride 0 0.018 0.025 0.05 Lipoic acid 0 0.12 0.25 0.5 Choline chloride 0 0.014 0.03 0.06 Serum replacement Basic culture medium Supplemented medium X Supplemented medium Y Supplemented medium Z Insulin 20 17 10 10 Transferrin 20 18 10 10 Cholesterol 50 100 150 200 Bovine serum albumin 35 50 75 100 Lipid mixture 0 0.15%v/v 0.25%v/v 0.5%v/v Primatone 0 1800 2500 5000 Polyethylene glycol 0 0.032 0.04 0.08

。

3. Use of the set of media of any one of claims 1-2 for transient expression of a foreign protein by HEK293 cells in vitro.

4. The use according to claim 3, wherein the foreign protein is a VEGF protein or an M protein of Zika virus.

5. Use of the set of media of any one of claims 1-2 for viral packaging of HEK293 cells in vitro.

6. The use according to claim 5, wherein the virus is a recombinant adenovirus expressing classical swine fever virus E2 protein.

7. A method for culturing HEK293 cells, which comprises using the culture medium set of any one of claims 1-2, resuscitating HEK293 cells in a basal medium, replacing the cells with a feed medium X after 12 hours, subculturing the cells for 36 hours, subculturing the cells with a feed medium Y, and subculturing the cells for 36 hours, wherein the seeding density of the cells for each subculturing is 1-2X 10⁶/ml。

8. The HEK293 cell culture method according to claim 7, which comprises the following specific steps:

step 1: recovering the frozen HEK293 cells in a basal medium at 37 ℃ and 5% CO₂Resuscitating the cells in the basal medium of the culture medium set according to any of claims 1-2 to maintain the state of the cells;

step 2: counting the cells, observing the cell viability, changing to a supplemented medium X when the recovery viability reaches above 70% and/or the culture time reaches 12h, adapting the cells to the culture environment, and continuing to culture the calculated cellsThe survival rate is that when the survival rate is more than 95 percent and the cells are judged to be in the logarithmic phase, and/or after the continuous culture time is 36 hours, the supplemented medium Y is started to be used, the continuous culture is carried out for 36 hours, the supplemented medium Z is used, the cells are ensured to be always in the logarithmic phase, and the inoculation density is 1-2 multiplied by 10⁶/ml；

And step 3: when the cell density reaches 3-4X 10⁶When the cells are cultured at 3X 10⁶Inoculating the culture solution into a six-well plate at a density of/ml for overnight culture;

and 4, step 4: preparing 1-2mg/ml of transfection plasmid and 2-4mg/ml of transfection reagent PEI, diluting the transfection plasmid by using a supplemented medium Z, uniformly mixing by using a vortex oscillator, adding into a six-hole plate, and supplementing the supplemented medium Z for culturing;